The invention is in the area of screening assays for identifying compounds that can affect gene expression, and specifically for identifying compounds that affect interactions between RNA and RNA binding proteins.
The regulation of protein expression can occur at a number of levels: transcriptional, post-transcriptional, or post-translational. The modulation of protein expression is often critical for the treatment of disease. Recent work at modulating protein levels by altering transcriptional activity has resulted in preclinical research programs being established and licensing agreements being entered into. For example, Ligand Pharmaceuticals, Inc. (San Diego, Calif.) has entered into multiple drug discovery programs with large pharmaceutical companies based on their Signal Transducers and Activators of Transcription technology for use as anti-inflammatory, anti-cancer and hormone replacement therapies. In addition, Oncogene Science, Inc. (Uniondale, N.Y.) is using its proprietary gene transcriptional technologies to develop biopharmaceutical products for the treatment of cancer. Other companies, such as Signal Pharmaceuticals, Inc. (San Diego, Calif.) and Tularik, Inc. (San Francisco, Calif.) are developing small molecules that regulate transcription factors. While this approach holds promise, no compounds have yet to make it to clinical trials. The lack of specificity of transcription factors and requirement for nuclear localization are two concerns with this technology. In the first case, a drug affecting the binding of a transcription factor may affect transcription of many genes other than the target gene. In the second case, it is difficult to design a drug that both has the proper interaction with a targeted transcription factor and is transported into the nucleus where it exerts its effect. Inhibition of protein expression by targeting the RNA is an alternate approach involving antisense technology. The antisense technology has also generated much interest with several products in clinical trials (ISIS2105, ISIS2922 and ISIS2302). However, the major drawbacks with this approach are the cost of oligonucleotides, the ability to deliver the oligonucleotides into cells, and their inability to increase protein levels.
A major area of post-transcriptional regulation in eukaryotic cells involves the specific interaction of proteins with RNA. These RNA binding proteins (RBP) appear to mediate the processing of pre-mRNAs, the transport of mRNA from the nucleus to the cytoplasm, mRNA stabilization, the translational efficiency of mRNA, and the sequestration of some mRNAs. Recent studies have identified several RNA-binding motifs in a diversity of RBPs. The most common RNA binding protein motifs are the RNP motif, Arg-rich motif, RGG box, KH motif and double-stranded RNA-binding motif (for review see Burd and Dreyfuss, Science 265:615-621 (1994)). These motifs recognize both sequence and structure dependent RNA elements. In the case of the double-stranded RNA-binding motif, sequence recognition is unimportant. However, in addition to the double stranded structure, a positional effect for the double-stranded RNA may play a role in recognition (Bass, Nucleic Acids Symposium 33:13-15 (1995)) and some of these proteins may also require binding to Z-DNA prior to their activity on the double-stranded RNA (Herbert et al., Proc. Natl. Acad. Sci. USA 92:7550-7554 (1995)). In addition, other RNA binding proteins, such as AUBF (Malter, Science 246:664-666 (1989)) are likely to bind in a structure-independent manner.
Due to the clear importance of RNA/RBP interactions in the regulation of gene expression, these interactions would be an attractive target for drugs that affect them for modulation of protein levels in disease states. To fully exploit these interactions as therapeutic targets, however, requires a clear understanding of how these interactions affect expression, which RBPs are involved in the regulation of RNAs of interest, and the ability to study the modulating effects of potential drugs on the RNA/RBP interactions. To fully exploit such interactions also requires identification of binding sites for RBPs in RNA molecules of interest.
Many investigators have used mobility shift assays to detect RNA/protein interactions. However, the conditions established in one laboratory often fail to detect interactions of different molecules. In addition, the diversity of RNA structures and binding motifs in the protein have led numerous investigators to conclude that a single set of conditions would be impossible to define for detection of multiple different interactions. With more genes being identified as being post-transcriptionally regulated, a universal set of binding conditions would allow for the detection and characterization of the molecules involved in these interactions and ultimately would provide targets for which therapeutics could be developed. Such universal assay conditions have been described in PCT application WO 98/04923.
Therefore, it is an object of the invention to provide an assay for the identification of compounds that affect the interaction of binding sites for RNA binding proteins.
It is a further object of the invention to provide nucleic acid sequences which interact with RNA binding proteins.
Disclosed are nucleic acid sequences which, when present in an RNA molecule, bind to RNA binding proteins. These nucleic acid sequences are present in untranslated regions of certain mRNA. They can be used in assays to identify compounds that affect the interaction of RNA containing the nucleic acid sequence, such as the source mRNA, and RNA binding proteins. The disclosed nucleic acid sequences can also be used to identify RNA binding proteins that can interact with the sequences.
An assay for identifying compounds that affect interaction of RNA containing one of the disclosed nucleic acid sequences and RNA binding proteins is also disclosed. The assay involves detecting interactions between RNA binding proteins and an RNA molecule containing one of the disclosed nucleic acid sequences in the presence of a test compound and in the absence of the test compound. A difference in the detected interaction in the presence and absence of the test compound indicates that the compound affects the interaction. For example, the assay can be done by forming a test solution and a control solution that each include an RNA molecule containing one of the disclosed nucleic acid sequences, heating the test solution and control solution to denature the RNA molecule, cooling the test solution and the control solution, adding a test compound to the test solution, adding an RNA binding protein to the test solution and the control solution, and detecting interactions between the RNA molecule and the RNA binding protein in the test solution and the control solution. A test compound is identified as a compound having an effect on interactions between the RNA molecule and the RNA binding protein if the interactions detected in the control solution and the interactions detected in the test solution containing the test compound differ.
The identified compounds can be used to affect the interaction of RNA binding proteins with mRNA containing the nucleic acid sequences or related sequences. This can alter expression of the mRNA since a major area of regulation of gene expression involves the regulatory effect of RNA binding proteins interacting with RNA molecules. Interactions between RNA molecules and RNA binding proteins are known to be involved in RNA stabilization, translational efficiency, RNA localization, RNA transcription, RNA editing, and RNA splicing and the identified compounds can be used to affect these processes.